A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector.
A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector.
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DOI：
10.1371/journal.pone.0017267
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发表时间：
2011-02-24
期刊：
影响因子：
3.7
通讯作者：
Ohbayashi T
中科院分区：
文献类型：
--
作者：
Yamaguchi S;Kazuki Y;Nakayama Y;Nanba E;Oshimura M;Ohbayashi T
能够表达目的基因的细胞的产生对于生物医学和生物技术中的多种应用是重要的，包括基因治疗和动物转基因。使用位点特异性重组酶如Cre、FLP和ΦC31在基因组中的精确位置插入转基因的能力对于转基因效率具有主要益处。最近对来自ΦC31、R4、TP 901 -1和Bxb 1整合酶的研究表明，这些重组酶催化哺乳动物细胞中的位点特异性重组。在本研究中，我们研究了整合酶在哺乳动物细胞中的位点特异性重组和基因表达的活性。我们设计了一个含有5个重组位点（ΦC31 attP、R4 attP、TP 901 -1 attP、Bxb 1 attP和FRT;多整合酶HAC载体）和从头哺乳动物密码子优化的整合酶的人人工染色体（HAC）载体。多整合酶HAC载体具有多种功能，包括在精确基因座中的基因整合和避免基因组位置效应;因此，它被用作研究整合酶活性的平台。整合酶以39.3- 96.8%的频率进行位点特异性重组。此外，我们在使用多整合酶HAC载体获得的77.3-87.5%的集落中观察到同源基因表达。该载体也可转移到另一种细胞系，并且能够在该环境中接受感兴趣的基因。这些数据表明，整合酶在哺乳动物细胞中具有高DNA重组效率。多整合酶HAC载体使我们能够有效地产生转基因表达细胞，并创建用于基因表达的平台细胞系。
The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3–96.8%. Additionally, we observed homogenous gene expression in 77.3–87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.
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影响因子：
3.5
作者：
Koduri, RK;Miller, JT;Thammana, P
通讯作者：
Thammana, P
影响因子：
14.9
作者：
GRANTHAM, R;GAUTIER, C;GOUY, M
通讯作者：
GOUY, M
影响因子：
5.1
作者：
Kakeda, M;Hiratsuka, M;Tomizuka, K
通讯作者：
Tomizuka, K
影响因子：
46.9
作者：
Hirata, R;Chamberlain, J;Russell, DW
通讯作者：
Russell, DW
DOI：
10.1073/pnas.0504505102
发表时间：
2006-01-10
影响因子：
11.1
作者：
Bertoni, C;Jarrahian, S;Rando, TA
通讯作者：
Rando, TA